The dimer of chimeric recombinant binding domain(B)-functional group(F) fusion was the first to be formed by using disulfide-bond-bridge to connect monomers having twice number of binding domain, and it has higher efficiency for targeting its functional group to the targets than the monomer and the production yield is high containing said extension peptide chain(LFA, lath flexible amino acid).
The fusions of binding domain and heterogeneous functional group have been made of various kinds of binding domains and heterogeneous functional groups.
Antibody is a typical use of binding domain[Reference: Hall, Walter A., Immunotoxin Method and Protocols, Method in Molecular Biology Vol 166, Humana Press, Totowa, N.J.]. Antibody has been studied with changing its binding region through recombination and modification maintaining its binding affinity and binding specificity. For examples, there are scFv, pFv, dsFv, Fab, L(using one light chain), LL(using two light chains), H(using one heavy chain), HH(using two heavy chains), diabody, triabody, tetrabody, double headed antibody and others[Reference: Brinkmann, U., et al., J. Mol. Biol. 268, 107˜117, 1997, Chaudbary, V. K., et al., Nature 339, 394˜397, 1989, Webber, K. O., et al., Mol. Immunol. 4, 249˜258, 1995, Yokota, T., et al., Cancer Res. 52, 3402˜3408, 1992, Kreitman R. J., et al., Leukemia 7(4), 553˜562, 1993, Pluckthun A. and Pack P., Immunotechnology 3, 83, 1997, Hollinger, P., et al., Protein Eng. 9, 299˜305, 1996, Atwell J., et al., Protein Eng. 12, 597˜604, 1999, Iliades P., et al., FEBS Lett. 409, 437, 1997]. These fragments of antibody binding regions have been used independently without fusing to functional groups or used as a fusion with functional groups which provokes physiological responses to target cells to deliver the functional group specifically.
Also, examples for use as binding domain are, many kinds of ligands or fragments which have ligand binding affinity, for example, TGF alpha, TGF beta, IL2, IL6, TNF, GMSCF and more. And they include many kinds of ligand receptors or fragments which have receptor binding affinity, for example, TBP1, TBP2, IFN alpha or beta receptor, gonadotropin receptor and other receptors.
There are many functional groups that have been used in fusion with binding domains [Reference: Hall, Walter A., Immunotoxin Method and Protocols, Method in Molecular Biology Vol 166, Humana Press, Totowa, N.J.]. For example, enzymes that have functions in prodrug transformation, material detection, decomposition, formation, proteins containing cytotoxic functional group and other functional group, organisms including the viruses for gene therapy, compounds that form cationic tail for delivering DNA, drug compounds, liposomes for drug delivery, biosensors for detecting real time target molecule and many others are used as functional group for fusion[Reference: (Hudson, P. J., Curr Opin Immunol 11(5), 548˜5, 1999)(Bagshawe, K. D., et al., Curr Opin Immunol 11(5), 579˜83, 1999)].
Antibody-toxin functional group fusion is a molecule which has cytotoxic factor connected via chemical or genetic method with antibodies as specific cell binding-domain [Reference: Cobb, P. W., et al., Semin Hematol 29, 6˜13, 1992]. Antibody-toxin functional group fusion was expected to be successful in cancer treatment after the development of the antibody recognizing cancer cell.
Antibody-toxin functional group fusion in its early stages was made by connecting two proteins via protein chemical cross-linking reaction, but in accordance with the development of recombinant DNA technology, it was produced in various forms of recombinant protein through genetic fusion. The incipient of antibody-toxin fusion(mAb-toxin) made by protein chemical cross-linking showed high stability of the fusion protein in blood and it exterminated the cancer cells at clinical demonstration[Reference: Pai, L. H., et al., Cancer Res. 52, 3189˜93, 1992] but, the damages on antibody caused during chemical reaction and inactivate molecules produced by chemical side reactions remained as problems.
These problems were solved mostly through recombinant DNA technology. Genetic engineering allowed genetically fusing the essential elements for antibody-toxin fusion to make molecules in purity and homogenously, and also it allowed small molecular weighted-proteins to be designed and produced[Reference: Pai, L. H., et al., Proc Natl Acad Sci USA 88, 3358˜3362, 1991]. For the minimum domain for antibody-toxin functional group fusion, the variable region of the antibody for binding (except constant region) and toxic enzymatic region of toxin (except cell-binding domain of toxin) [Reference: Kondo, T., et al., J Biol Chem 263, 9470˜9475, 1988] was used. However, nowadays binding domains and toxic domains themselves are modified to be made as derivatives for better activity[Reference: (Pastan, I., et al., Science 254, 1173˜1177, 1991)(Pastan, I., et al., Proc Natl Acad Sci USA 88, 3358˜3362, 1991)(Vitetta, E. S., et al., Cell Biology 2, 47˜58, 1991) (Allured, V. S., et al., Proc. Natl. Acad. Sci. USA 83, 1320˜1324, 1986) (Hwang, J et al., Cell 48, 129˜136, 1987)].
The modified antibody binding region produced by genetic recombination can be classified in 4 types. These are scFv(single chain Fv form) characterized in connecting the minimum binding unit of antibody VH and VL with 15 amino acid polypeptide linker (Gly4Ser4) [Reference: Buchner, J., et al., Anal Biochem 205, 263˜70, 1992], dsFv(disulfide-stabilized Fv form) characterized by connecting VH and VL via disulfide bonds, pFv(permutated Fv form) characterized in connecting VH and VL with base loop and Fab form etc. scFv-toxin functional group form has the smallest molecular weight from antibody binding domain produced and for this it was expected to have good penetration ability into cancer tissues showing good cytotoxicity. However, the low productions yield[Reference: (Buchner, J., et al., Anal Biochem 205, 263˜70, 1992)(Brinkmann. U., et al., Proc. Natl. Acad. Sci. USA 88, 8616˜8620, 1991)] and short half life in animal blood circulation[Reference: Brinkmann. U., et al., Proc. Natl. Acad. Sci. USA 89, 3065˜3069, 1992] were problems and there were no effects observed from results of clinical demonstration.
dsFv-toxin functional group has similar size with scFvtoxin functional group and made to have high stability in animal blood circulation. This type of antibody-toxin functional group was more stable than scFv toxin in blood circulation but the cytotoxicity test with cultured cell in vitro showed similar results[Reference: (Pastan, I., et al., Science 254, 1173˜1177, 1991) (Pastan, I., et al., Cancer Research 51, 3781˜3787, 1991)]. Results on dsFv distribution tests in animal with radionuclide labeled dsFv-toxin functional group[Reference: Choi, C., et al., Cancer Res. 55, 5323˜9, 1995] showed that dsFv disappeared from the blood circulation through excretion more fast than to bind with cancer cells and accumulate.
pFv is made by connecting the β-strand between 3 and 3b on VL and β-strand between 3 and 3b on VH[Reference: Brinkmann, U., et al., J Mol Biol 268, 107˜17, 1997]. However, this form of antibody-toxin functional group showed short half-life rate, low production yield, and no improved cytotoxicity effect.
Recombinant Fab-toxin functional group fusion was made to overcome the problem mentioned above[Reference (Ghetie, M. A., et al., 1991)(Kreitman, R. J., et al., Cancer Res 53, 819˜25, 1993)(Choe, M., et al., Cancer Res 54,3406˜7, 1994) (Kreitman, R. J et al., Int J Cancer 57,856˜64, 1994)]. This molecule showed similar half life of activity in blood circulation as the incipient antibody-toxin functional group chemical fusion(mAb-toxin) although it was a recombinant antibody-toxin functional group fusion and was more stable than scFv-toxin functional group, dsFv toxin functional group, pFv-toxin functional group in structure[Reference: (Choe, M., et al., Cancer Res. 54, 3460˜7, 1994)(Kreitman, R. J., et al., Int J Cancer 57, 856˜64, 1994)]. Also, the production yield of refolding was 10 times higher in maximum[Reference: (Buchner, J., et al., Bio/Technology 9, 157˜162, 1991)(Buchner, J., et al., Anal Biochem 205, 263˜70, 1992)]. Even though the Fab-toxin functional group in blood circulation was more active than scFv, the therapeutic efficacy towards cancer cells in animal model was good or had no big difference according to antibody type. The reason it had no big difference was assumed to be that Fab-toxin functional group has a bigger binding domain that each of the quaternary structure of Fab didn't form properly during refolding or it has two more intrachain disulfide bond to make disulfide bond incomplete or make disulfide bond scrambled or the cysteins for interchain disulfide bond between heavy and light chain did not form disulfide bond completely or got scrambled. Therefore, the complete structure formation of molecule was disturbed to have abnormal binding affinity against antigen and these may have mixed with the normal molecules to lower the efficacy[Reference: Choe, M., et al., Cancer Res 54. 3460˜7, 1994].
In this way, the Fab-toxin functional group has weak binding affinity because the isolation and removal of the inactivated molecules and molecules with disulfide bond scrambled or incomplete formed is very difficult. According to these results, the recombinant antibody-toxin functional groups with the best stability in structure and appropriate half-life are Fab-toxin functional group form. To explain this biochemically, CH1 and CL and the disulfide bond between light and heavy chain give high stability to quaternary structure, and the resistance against degradation and clearance will be strong. The most important issue of the antibody-toxin functional group is stability and affinity.
Therefore, development of new antibody-toxin functional group is needed with improved stability and affinity to have high efficacy for targets and high productivity. To increase structural stability, the number of disulfide bond can be increased but if wrong bonds form or scrambling happen molecules with inactive structures are produced and the production yield lowers or doesn't produce it at all.
In addition to the described molecules mentioned above, there are toxin functional group-antibody fusions, which have toxin functional group on the amino terminal and antibody binding domain on the carboxyl terminal and they showed similar results.
Also, antibody derivatives with multiple binding domains were produced to increase binding affinity and they are diabody, triabody, tetrabody, double headed antibody and others[Reference: Takemura S., et al., Cancer Immunol Immunother. 51(1): 33˜44, 2002]. Increasing the numbers of binding domains two, three, four times and with the affinities between the chains composing binding domains, multiple binding domain derivatives are made. They showed that the binding affinity increased by the increase of binding domain numbers and there was no report before about these molecules being fused with heterogeneous functional groups to produce chimeric fusion. It is a difficult matter to connect binding domain to functional group without hindrance to each other and between the functional group themselves in forming dimeric form of the chimeric fusion and manufacturers concerned will know that this is the key to successful production. If the extension chain, which connects binding domain and functional group, disturbs the refolding of the big chains of binding domain or functional group or some hindrance occurs between the big chains, no production will occur.
Also, when the Cysteine lacking intrachain disulfide bond counterpart cysteine is added in the extension sequence connecting binding domain and functional group this may form disulfide bond with other Cysteine, which has its right counterpart, and this may lead to disulfide bond structure scrambling. In this case the uncoupled Cysteine that has no natural disulfide bond counterpart will form wrong disulfide bonds to ruin sterical structure of the molecule and lose its activity. And also, it is natural to think that if the extension chain containing uncoupled Cysteine has lots of flexible amino acids to be long making the extension chain have no regular structure, it is easier for the uncoupled Cysteine to intermix with other naturally coupled Cysteine in neighboring big structure. The inventors experienced these kinds of failures in producing active dimer molecules and it will be thought as a special and incidental case when the manufacturers concerned produce one of these types of molecule.
If the problems and limits mentioned above are overcome by new findings about forming dimers with disulfide bond generally, the dimer of binding domain(B)-functional group(F) fusion by disulfide bond will be possible by the manufacturers concerned.
On the other side, there were reports about compared studies of various structures of molecules[Reference: Bera, T. K., et al., Bioconjug Chem 9(6), 736˜4, 1998] but no reports were found about producing dimer[antibody-toxin functional group]2 by using disulfide bonding between antibody-toxin functional groups fusion to form molecules with double binding valency. Therefore, it has been expected that the manufacturers will be able to know how extension peptide chain to be put in between big sequences of antibody and toxin functional group and how the uncoupled Cysteine to be put in to extension peptide chain to form disulfide bonds for dimerization after the understanding about the refolding process forming the tertiary and quaternary structure of the protein.